Cancer continues to be a serious illness even though it has been a center of research for a long time. Tumors can be classified as benign (e.g., non-cancerous and normally don't spread) or malignant (e.g., cancerous that can spread to different parts of the body). Cancer can spread to different parts of the body via two different mechanisms: (1) invasion; and (2) metastasis. Invasion involves direct migration and penetration by cancer cells into adjacent tissues, whereas metastasis refers to cancer cells penetrating into circulation (e.g., lymphatic and blood vessels) followed by invasion of normal tissue somewhere else in the body. That is, metastasis results in additional secondary tumors away from the original, primary tumor location. This allows cancer to spread to almost anywhere in the body. The complex process of metastasis can be broken into four individual steps: (1) Breakup of tumor cells from original site and migration into the circulatory system; (2) Attachment of circulating tumor cells to the vessel walls; (3) Migration of tumor cells from vessel into tissue; and (4) Invasion of tumor cells forming new colonies.
Metastatic cells break away from the other cells in the tumor, and overcome constraints on cell movement imposed by the basement membrane and other barriers. Tumor cells often secrete proteases that help the tumor cells digest the basement membrane and invade into the extracellular matrix, which can be followed by release into circulation. Commonly, the ability of a tumor cell to invade the matrix is taken to be indicative of its metastatic nature. However, the process of cancer metastasization in vivo is more complex and only a few of the tumor cells form metastatic tumors. In order to metastasize, the cells first have to survive circulation, adhere to the vessel wall and migrate/invade into normal tissue to form new colonies. Usually, the host immune response or high shear zones are likely to destroy tumor cells in circulation. Furthermore, only a fraction of the circulating metastatic cells adhere to the vessel wall, potentially using several different mechanisms. For example, studies show that these tumor cells in the circulation often utilize the presence of leukocytes to enhance adhesion and extravasation. Cell adhesion prior to migration is dependent upon the microenvironment including geometric features of the vasculature and the associated local hemodynamic factors such as wall shear stress, dynamic pressure, and residence time in circulation. Thus, accurate characterization of metastasis involves representation of adhesion and circulation, in addition to migration or invasion.
Adhesion of tumor cells to the endothelium is hypothesized to occur by two potentially very different mechanisms. According to the first hypothesis, tumor cells are trapped in capillaries based on vessel-size restriction. The second theory argues that tumor cells can adhere to endothelium by forming shear-resistant bonds with the endothelium. Basic cellular adhesion process has been well studied over the past two to three decades for the development of both static and fluidic assays. The process of adhesion involves rolling of cells on the endothelium, mediated by selectins, followed by a firm adhesion process mediated by integrins.
Previously, researchers have been studying particle adhesion on the endothelium using idealized straight channels under fluidic conditions. However, these devices lack correspondence with in vivo geometry, scale/aspect ratios (e.g., microvasculature vs. large vessel models), large reagent volumes, and are inadequate in studying adhesion event differences between healthy and diseased vasculature.
After a tumor cell has adhered to the walls of the endothelium, it can start the process of migration/invasion into the tissue using gaps in the endothelium. There are currently several in vitro methods that study cell migration that can be classified as 2-D or 3-D cell migration models. 2-D models are easy to set up and run, but do not represent the microenvironment of living tissues and the migration is unidirectional. 3-D models, on the other hand, are difficult to set up, but can be used to mimic the microenvironment using extracellular matrix components. The classical way to study 2-D cell migration is the use of a two-chamber system commonly known as the Boyden chamber or the transwell assay. It consists of a porous membrane separating two chambers and the cell migration from one chamber to another in the presence of a chemical signal (chemoattractant). Several companies have adapted this format for high throughput analysis using 96 and 384 well plate formats. A major problem with the Boyden chamber is the inability to visualize cells during migration. In addition, the end point measurement involves staining procedures, which are tedious and allow for inaccurate assessment of the migrated cells.
Recent modifications to this technique such as the Dunn chamber have allowed direct viewing of the cell migration but have also posed new problems with the study being performed over long annular (1 mm) distance. Incorporation of shear flow in these filter assays, while possible, is inefficient due to the large reagent volumes needed. The large fluid volumes also render the system inaccurate as they cause membrane fluctuation, which leads to strong disturbances in set shear conditions.
A recent entry to the field, μ-Slide chemotaxis assay from Ibidi, LLC (Verona, Wis.) consists of linear channels allowing real-time microscopy. However, the migration takes place under static conditions and cells migrate along a linear path with no obstructions (filters, etc.). In addition this device is a 2-D device and is not amenable to studies on suspension cells. From among all these devices, the Boyden chamber is still the most commonly used device.
Therefore, there remains a need in the art for a better system and methodology for studying tumor metastasis.